QUICK MANUAL FOR USING THE LEICA APPLICATION SOFTWARE (LAS) INSTALLED ON THE COOLLEI COMPUTER

Leica camera DFC 420C (SN384681109)

Room HG01.235General Instrumentation - FNWIRadboudUniversity

For (full) instructions to new users or other questions, please contact the assistant
(see also Leica LAS Help menu):

Liesbeth Pierson, Tel 024-3652012, room HG 01.222,

Email

Or if absent, Geert-Jan Janssen or Jelle Eygensteyn

Website: and and then follow the menu under light microscopy

  1. GETTING STARTED more…
  2. CHOOSING BINOCULAR OR ORTHOPLAN SETTINGSmore…
  3. STORING CAPTURED IMAGES/MOVIESmore…
  4. IMAGE OR MOVIESmore…
  5. MAIN FUNCTIONS FOR IMAGE ACQUISITIONmore…
  6. THUMBNAILS, BROWSE, PROCESSmore…
  7. CLOSINGmore…

A. GETTING STARTED

  1. Ask assistance to get instructions and to have the camera connected to the desired device (binocular or microscope)
  2. Start Windows (no password required)

  3. To preview or capture images with the camera, open the LAS software by double clicking on the LAS icon.

B. CHOOSING BINOCULARORORTHOPLAN SETTINGS



  1. Magnification calibrations have been pre-entered for the two most useddevices:
    a) The Leica MZFLIII binocular. Its starting menu looks like this:
    b) The Leitz Orthoplan microscope. Its starting menu looks like this:
    If the device of your current choice is already the one appearing upon opening the software, (or if another device that the other pre-registered device is required. Note that in this case magnification settings indicated by the software will be erroneous), proceed to item 10.
    If theother pre-entered device is required, go to File> Exit LAS

    To “Are you sure you want to Exit LAS?”, answer “Yes”
  2. A new menu opens. Under Options, choose Hardware Setup, like in:

  3. Please select the connected microscope from the list, either:
    a) Leica MZ12.5 for the binocular, exactly like shown here (so do not choose
    X/Y stage or other items) and press Save

orb) the Leica generic microscope and Orthoplan for the microscope, and press Save.

DO NOT CHANGE ANY OTHER SETTINGS!

  1. After having changed a device choice, go to File> Exit and confirm exit by “Yes”

  2. Restart LAS software. Now the desired device setup should appear. (In case of troubles, contact Liesbeth or back-up administrator Geert-Jan)
  3. Always check the calibrations, for example with an objectmicrometer, because errors might be introduced automatically. Photoshop calibration tables for the Orthoplan and the binocular are available on the Coolei pc desktop and on the GI server (geminstr-srv.science.ru.nl, login with your cncz name and password)
    \CLSM-&-Lightmicroscopes\A-Calibration-Magnification-LightMicroscopes\LeicaDMIRE2-CLSM).

C. STORING CAPTURED IMAGES/MOVIES

  1. To save images, create (if not done already) with Explorer an own folder under E:>DATA>Users>the own Department> and your name (first and last name), e.g. E:DATA>Users>General Instrumentation>Pietje Bel
    where captured images/movies will be temporarily stored
  2. In order to record captured images/movies in the own directory as default path, go to “Browse”, click on the desired path (DATA (E) >Your department>Your name> own sub folders,if you like, and then press the “Set capture location “ button.
    The newly defined default path for storing images is indicated in red.
    You can retrieve data from this folder and copy them to an own storage stick, server or burn them on CD/DVD.

After a session, PLEASE, copy the captured images to your own storage station within one week. Check that all data have been correctly transferred and delete the original data from the Coollei PC yourself to prevent clogging. There will be frequent clean-up actions for files older than one week. If for any reason a longer storage time is required, contact Liesbeth
D. IMAGE OR MOVIES

  1. For switching between image acquisition to acquiring movies or time-lapse series (=Multi-time) go to “Select Acquisition Mode”:
    and click the desired modus.

    E. MAIN FUNCTIONS FORIMAGE ACQUISITION
  2. Overview (details furtheron)
    aautomatic exposure
    bautomatic white balance
    cpop-up menu camera settingssummarizing most functions
    dis inactive
    e Exposure Adjust is to set parameters for acquiring an image
    f Input Options resolution life/captured images
    g Histogram distribution of pixel values
    h Processing shading correction, sharpening, flip

i Regions of interest for crop, local focus, zoom

j Calibration settings x pixels = y µm
k Scale bar size and appearance
l Linking inactive
m Check color hue and saturation corrections
n Acquire image to capture a view

Details of menu

  1. ad a Automatic exposure. Click once to set
  2. adbautomatic white balance
    Applies automatically white balance on full preview image
  3. ad c pop-up menu camera settingssummarizing most functions (see also details step by step here below)

  1. add inactive
  1. ad e Exposure: exposure time, Saturation (color intensity), gamma (linear or not), gain (for strong/weak signal)
  1. ad fInput OptionsDo not modify configure camera, keep cooling of the camera on.
    Recommended Live Format (preview) 864 x 648 Progressive large for bright field (quite rapid refreshing rate and still reasonable screen resolution; Progressive medium is more desirable for rapidly moving objects, the slower HG options when details must be considered for the focus)
    Recommended captured Format (final image after acquisition)are 1728 x 1296 Interlaced medium HQ or interlaced large HG (for some special applications, e.g. posters where the objective is not the bottle neck).
    For very weakly stained fluorescence samples (susceptible to bleaching) Color binning in both Live and Capture mode may be an outcome (poor resolution, but high sensitivity)
    Standard for imaging is 8 bits/channel. 16 bits per channel (effective less, 10 bits, I guess) is more suitable for semi-quantification (large dynamic range; can be opened in Photoshop and Image J)
  1. ad g Histogram shows the distribution of pixel intensities (over and under exposures)
  1. ad h Processing includes shading correction, sharpening (in general not recommended on the acquisition of original scientific images; sharpening is a typical post-acquisition processing, eg to apply with Photoshop) and image flipping. Shading correction is useful when the background illumination is uneven or when a spot of dirt on the target of the camera obscures the view. First, create a shading reference, without the object (possibly slightly out of focus), but with all characteristics for a certain objective or zoom, illumination profile and orientation of the camera. Take care that no shading settings of earlier images/session are inappropriately still active. Choose then None.
  1. ad i Regions of interest crop to ROI, white balance, spot exposure spot focus spot zoom (preview)
  2. Automatical White Balance
  3. How to switch on/off region of interest? (E.g. to determine the best focus)
  1. ad j Calibration settingsaccording to pre-entered calculations. Control whether OK!

  2. ad k Scale bars adjustments
    Calibration images have been made and compiled to provide scale bars in a Photoshop document in separate layers for the various settings (see GI server under CLSM-Light microscopy then calibrations). However, always check yourself that the settings are correct, using an object micrometer (ask assistant for it).
  3. ad l Linking not active
  4. ad m Check color to manually correct color by dragging the cursor. Drag a region (can be resized) over the life image and click check color. A dot and a cross appear. Shift the cross to adjust the color balance. The dot shows the result. In an equally balanced RGB signal (e.g gray) the dot appears in the center.
  5. ad n Acquire image (image and encrypted calibration settings will be stored. However, note which objective was used or what the zoom factor was for later size calibrations)

F. THUMBNAILS, BROWSE, PROCESS

  1. To see thumbnails of already captured images activate the icon indicated in blue here
    To switch between the image size indication and the frame refresh rate, press control + R (indicated in green)
  2. The Browse menu allows on to view images under various zoom, to navigate within a zoomed image, to view information under i.

  3. Functions of the Process menu

G. CLOSING

Go to File Exit. (Captured images can be retrieved from the Users folder)

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